Flavoproteins catalyze many biologically important electron- transfer reactions. We have developed and used a spectroelectrochemical method to systematically study the thermodynamic properties of two flavoprotein groups, the oxidases and the dehydrogenases. We plan to finish the characterization of the three main groups of flavoproteins by studying the monooxygenases, and we propose to extensively study the ligand binding properties of the flavoprotein dehydrogenase enzymes involved in the beta-oxidation of fatty acids. A parallel study will be performed on ligand binding to the monooxygenases. The beta-oxidation of fatty acids is an important energy conversion process in heart muscle, skeletal muscle, liver and kidney. It is the primary energy source for heart muscle, providing 90% of this energy. The beta-oxidation process is regulated by the acyl-CoA dehydrogenases (ACD), which catalyze the first step. It has been postulated that the ACD's are isopotential with their substrates and that product binding controls the completeness of the reaction. Using enzymes from a bacterial source we have shown that the substrate is not isopotential with the enzyme and that the binding of the substrate or product to the reduced form has shifted the potential significantly positive. A similar shift would be of more significance in a mammalian system. Work done by others suggests that such shifts are occurring. This work taken together strongly suggests that for the mammalian system, substrate-bound ACD is isopotential with electron transferring flavoprotein (ETF) and with ETF-ubiquinone oxidoreductase. This suggestion has profound implications on the energetics of the beta-oxidation process. This work suggests that a further redox study will give new inisights into regulation (and inhibition) of oxidation. Our aims are to measure the electron energy levels (redox potentials) of free enzymes and substrates, then to measure the effect of ligand binding on those levels. Thus, we will be able to assess the relative importance of binding in controlling catalytic reactions. Electrochemistry is the best method to examine these interactions because we can clearly show that electron-energy levels change in a measurable way upon ligand binding. We will perform a comparative study of acyl-CoA dehydrogenases, from mitochondria, bacteria which are mitochondrial precursors and anaerobic bacteria bound to substrates, products, inhibitors, and their respective ETF's.